The invention relates to cancer diagnosis.
Effective treatment for patients with cancers of the central nervous system (CNS) is, for the most part, an unmet challenge, and the incidence of both primary and metastatic brain tumors appears to be increasing. The prognosis of patients with malignant gliomas and brain metastases remains poor.
Cytological examination to detect malignant cells in the cerebrospinal fluid (CSF) is a standard method by which diseases such as leptomeningeal carcinomatosis are diagnosed. The absence of malignant cells is a primary endpoint for most therapeutic interventions. However, false negatives are often encountered. The inability to estimate or detect the residual burden of disseminated cells by conventional imaging technologies (computerized axial tomography (CT) scan or magnetic resonance imaging (MRI)) compromises decision making on prognosis and treatment.
The invention provides an accurate and reliable method of diagnosing primary brain tumors or brain metastases. A method for diagnosing a tumor in the CNS of a mammal is carried out by contacting a bodily fluid from the mammal with a detectable ITI ligand and measuring the amount of bound ITI ligand in the sample of bodily fluid. For example, the ligand is a monoclonal antibody or polyclonal antisera which binds to an ITI polypeptide, e.g., an ITI light chain polypeptide; a sample of bodily fluid is contacted with the antibody under conditions sufficient to form an antigen-antibody complex and the complex(es) detected. Alternatively, the ligand is a synthetic or proteolytically-generated peptide that binds to the ITI light chain. For detection purposes, the ligand, e.g., ITI-specific antibody, is directly or indirectly labelled using, e.g., a calorimetric or radioisotopic marker. The amount of the immune complex (which contains ITI antigen bound to ITI-specific antibody) is quantitated to determine the level of ITI in the fluid, and the level of ITI in the fluid is compared to a normal control level of ITI (e.g., a previously determined baseline value or the level of ITI from a subject known to be cancer-free). ITI is elevated in the bodily fluids of patients with CNS tumors as a result of secretion of ITI by disseminated cancer cells or as a result of breakdown of the ECM, which causes leakage of serum ITI into the CSF. Alternatively, tumor cells stimulate production of ITI by surrounding tissues in the brain such as choroid plexus and astrocytes. An elevated level of ITI compared to a normal control level indicates the presence of a tumor in the CNS of the tested mammal. For example, the presence of a tumor is indicated by an amount of ITI in a test sample which is at least two-fold greater than that in a normal control or normal baseline amount of ITI in a CNS bodily fluid such as (CSF). For example, the amount of ITI in the test sample is 2-7 fold greater than normal. The level of ITI in a CNS bodily fluid is indicative of a neurological tumor.
The bodily fluid is a preferably a CNS-derived fluid. By CNS is meant a bodily tissue of the brain or spinal cord. For example, a CNS-derived fluid is cerebrospinal fluid or the supernatant of a cell lysate of cells of the brain, spinal cord, or cells found in the CSF. Other bodily fluids such as blood, serum, urine, saliva, sputum, perspiration, lung effusion, or ascites fluid are tested for the level of ITI as an indication of CNS pathology.
Also with the invention is a method for prognosis of a tumor in the CNS of a mammal, which is carried out by (a) contacting a bodily fluid, e.g., a CNS-derived fluid, from the mammal with an antibody reactive with an ITI polypeptide, e.g., an ITI light chain, under conditions sufficient to form an antigen-antibody complex and detecting the antigen-antibody complex; (b) quantitating the amount of complex to determine the level of ITI in the fluid; and (c) comparing the level of ITI in the fluid with a normal control level of ITI. Changes in the severity of the disease are monitored by comparing changes in the level of ITI in bodily fluids of the patient over time. An increasing level of ITI, e.g., in the CSF, over time indicates an adverse prognosis, e.g,. increased CNS pathology. Such temporal data is used to determine a course of treatment for the patient.
The invention features a monoclonal antibody that binds to an epitope of ITI light chain. The antibody binds to residues within or proximal to an active site of the ITI light chain. When incubated in the presence of ITI and a serine protease, the antibody inhibits ITI-mediated inhibition of serine protease activity. Preferably, the antibody, e.g., mAb 69.31, does not bind to an octapeptide sequence of Urinary Trypsin Inhibitor fragment (UTI), Arg-Gly-Pro-Cys-Arg-Ala-Phe-Ile (SEQ ID NO:1).
Reagents, e.g., an ITI-specific antibody such as mAb 69.31, for carrying out the diagnostic or prognostic assay may be packaged together as a kit. For example, the antibody is immobilized on a solid phase and packaged together with other reagents suitable for detecting ITI-ligand complexes. For example, enzyme-conjugated reagents may be included; ITI may also be included as a standard or control reagent. The solid phase component of the kit onto which an antibody or antigen is immobilized is preferably an assay plate, an assay well, a nitrocellulose membrane, a bead, a dipstick, or a component of an elution column. For example, the capture antibody, e.g., mAb 69.31, is immobilized and a secondary antibody is used to detect the immune complex (e.g., an ITI antigen bound to the mAb 69.31 antibody. The kit may optionally contain a purified ITI polypeptide or purified ITI complex as a control. The polypeptide or complex is purified from natural sources or recombinantly produced. The kit may also contain a second antibody or other detectable marker. The second antibody or marker is labelled, e.g., using a radioisotope, fluorochrome, or other means of detection.
Also within the invention is a method of inhibiting metastases of a systemic cancer into the CNS of a mammal by administering to the mammal an ITI composition, e.g., an ITI polypeptide or a nucleic acid encoding an ITI polypeptide. For example, a sythetic oligonucleotide encoding an ITI polypeptide is administered. The ITI composition is administered by infusion directly into the CSF of the mammal. For therapeutic administration, the ITI polypeptide is administered in its native form as a molecular complex in which an ITI light chain is bound to an ITI heavy chain. The method may include a step of identifying a mammal at risk of developing a brain metastases. For example, the following cancers have a tendency to metastasize to the CNS, e.g, the brain: breast cancer, lung cancer, ovarian cancer, kidney cancer, malignant melanoma, esophageal cancer, head and neck cancers, testis cancer, choriocarcinoma, prostate cancer, bone cancers, and soft tissue sarcomas. Individuals diagnosed as having such cancers are among those at risk of developing a metastases of the cancer to the CNS. Individuals suspected of having CNS cancer are also identified by detecting clinical symptoms such as headache, nausea or vomiting, seizures, altered mental status, altered speech, visual abnormalities, and/or paralysis. A CT scan or MRI is often used to diagnose brain metastases. However, the assays of the invention provide significant advantages, e.g., increased sensitivity, over such conventional diagnostic tools. The claimed assays accurately and reliably detect the presence of tumor very early (prior to the time at which a tumor is detected by the appearance of physical symptoms or by CT or MRI).
A method of inhibiting metastases of a primary CNS cancer in a mammal is also within the invention. The therapy is carried out by administering to into a mammal an ITI polypeptide, e.g., by direct infusion into the CSF of the mammal. Individuals having been diagnosed with the following CNS cancer types are treated according to the invention: astrocytoma, glioma, or metastases into the brain from other cancer types such as leukemias or carcinomas.
Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.